Accuracy of yellow fever case definition of epidemiologic surveillance, São Paulo, 2018

ABSTRACT OBJECTIVE To evaluate the accuracy of yellow fever (YF) suspected case definitions from the Brazilian Ministry of Health (BMH) and World Health Organization (WHO), as well as propose and evaluate new definitions of suspected cases, considering confirmed and discarded cases. METHODS The retrospective study was conducted at the Instituto de Infectologia Emílio Ribas (IIER), using the Epidemiologic Surveillance Form of YF cases. From the confirmed and discarded cases of YF, a logistic regression model was developed. The independent variables were used in a proposed definition of a suspected case of YF and its accuracy was evaluated. RESULTS In total, 113 YF suspect cases were reported, with 78 confirmed (69.0%). The definitions by BMH and WHO presented low sensitivity, 59% and 53.8%, and reduced accuracy, 53.1% and 47.8%, respectively. Predictive factors for YF were thrombocytopenia, leukopenia, and elevation of transaminases greater than twice normal. The definition including individual with acute onset of fever, followed by elevation of ALT or AST greater than twice the reference value AND leukopenia OR thrombocytopenia presented high sensitivity (88.3%), specificity (62.9%), and the best accuracy (80.4%), as proposed in the model. CONCLUSION The YF suspected case definitions of the BMH and the WHO have low sensitivity. The inclusion of nonspecific laboratory tests increases the accuracy of YF definition.


INTRODUCTION
Yellow fever (YF) is an acute disease, endemic in some tropical areas of the Americas and Africa, and it can affect humans and non-human primates (NHP). It is caused by a virus of the genus Flavivirus, family Flaviviridae, and transmitted by mosquitoes. There are two cycles of transmission: sylvatic and urban. YF is a burden for public health due to its clinical severity and high potential for dissemination in urban areas 1 . Vaccination is the most important measure to prevent YF, especially its international spread. The World Health Organization (WHO) developed, by a coalition of partners (The Vaccine Alliance-GAVI and UNICEF) the comprehensive global strategy to eliminate yellow fever epidemics (EYE) (2017)(2018)(2019)(2020)(2021)(2022)(2023)(2024)(2025)(2026), to face the changing epidemiology of yellow fever, resurgence of mosquitoes and increased risk of urban outbreaks and international spread 2 . The last case of urban yellow fever in Brazil occurred in 1942, despite the sporadic record of cases of sylvatic YF. The disease was endemic until 1999, especially in the North and Central-West regions. Between 2000 and 2008, an expansion of a viral circulation was observed towards the East and South regions 3 .
YF is a notifiable disease in Brazil. Its definition of a suspect case is an individual with fever (up to 7 days) of sudden occurrence followed by jaundice and/or hemorrhagic manifestations, residing in (or coming from) a YF risk area, or in locations with confirmed epizootic in NHP or of isolation of virus in vector mosquitoes in the 15 days prior, not vaccinated against YF, or with ignored vaccine status 6 . In 2009, a study analyzed 28 confirmed cases of sylvatic YF in the State of São Paulo. However, only 50% of the cases matched the definition of suspect case 7 .
This study aimed to describe the suspect cases attended at the Instituto de Infectologia Emílio Ribas (IIER), to evaluate sensitivity and specificity of YF case definitions from the Brazilian Ministry of Health and WHO during the 2018 outbreak, and to propose highly accurate case definitions.

Study Design: Retrospective Observational Study
Data sources: data from patients with suspected YF treated at the IIER were collected using the Epidemiological Surveillance Forms, selecting sociodemographic, clinical, and laboratory variables. Most patients were from the State of São Paulo (96.2%), mostly from the municipality of Mairiporã (41.0%).
Inclusion requirement: suspect cases had a clinical sample collected for laboratorial etiological confirmation provided by Instituto Adolfo Lutz, Public Health Laboratory. Suspected cases of YF were confirmed or discarded by YF virus detection by real-time polymerase chain reaction (RT-PCR) technique (91% and 91.4%) or positive immunoglobulin M (IgM) serology (8% and 5.7%), respectively. Dependent variable: case classification (confirmed or discarded) Independent variables: sociodemographic, clinical (signs and symptoms), and laboratory tests.

Analysis Procedure
Characteristics and the confirmed and discarded cases for YF were evaluated by Pearson's chi-squared test and Fisher's exact test. Univariate analysis of signs, symptoms, and laboratorial alterations of suspect cases were performed in order to identify possible variables predictive of YF, considering odds ratio (OR) values. The variables with p-value ≤ 0.20 in non-adjusted analysis were considered for a logistic regression model to identify independent factors associated to YF. The final models were evaluated by Hosmer-Lemeshow test.
Receiver operating characteristic (ROC) curves were constructed for each of the explanatory models, and the predictive factors were considered to create proposed definitions (1 and 2). Those proposed definitions were evaluated by sensitivity, specificity, predictive value positive (PVP), predictive value negative (PVN), and accuracy, which were also used to evaluate the following official definitions of suspect case.

Brazil's Ministry of Health's -Brazil's definition (2017)
An individual with acute onset of fever (up to 7 days), followed by jaundice and/or hemorrhagic manifestations, residing in (or coming from) a risk area for YF, or from locations with occurrence of epizootic confirmed in NHP, or isolation of virus in vector mosquitoes, in the 15 days prior, not vaccinated against YF, or with ignored vaccine status. In cases of outbreak, it is recommended to adjust the definition of suspect case making it more sensitive for detection of the highest number of cases possible, taking in consideration the wide clinical spectrum of the disease 6 .

World Health Organization's -WHO definition (2015)
Any individual with acute onset of fever and jaundice occurrence within 14 days of onset of the first symptoms 8 .
For the analysis of both definitions (Brazil and WHO), a vaccinated individual was considered as one with immunization received in, at least, 10 days from the beginning of the symptoms 9 . Moreover, the cases with non-reported traveling to or residence in YF risk areas were included as possible exposition to sylvatic environments during the YF outbreak. Laboratorial value patterns considered to describe observed alterations were the following: thrombocytopenia with platelet count equal or under 150,000/mm3; leukopenia with leukocyte count equal or under 4,500/mm3; leukocytosis with leukocyte count equal or over 11,000/mm3; renal function alterations with serum urea values over 40mg/dL and/or creatinine over 1.3 mg/dL; hyperbilirubinemia with total bilirubin serum dosage over 2 mg/dL; and increase of twice the reference value for transaminases considering the maximum value of 40 U/L for alanine aminotransferase (ALT) and aspartate aminotransferase (AST).The significance level adopted was 5% for all hypothesis tests. Analyses were performed using SPSS for Windows v.25 and Stata/MP 14.0 for Windows software. The study used data from hospital epidemiological surveillance. The data was approved by the Ethics Committee of IIER (Protocol 024329/2018).

RESULTS
In total, 113 suspect cases of YF were reported at IIER from January to November 2018, with 78 confirmed cases (69.0%). Of these, 23 individuals died (lethality of 29.5%). Males predominated among confirmed (80.8%) and discarded (74.3%) cases, without statistical difference between both groups (p=0.436). Analysis of age group among suspected population showed a predominance of adult cases (from 18 to 59 years old) (77.9%) and similar distribution between confirmed and discarded (p = 0.061). Whites were prevalent among suspect cases (69.9%), without statistical difference among confirmed and discarded (p = 0.183). Educational level was not different between those groups (p = 0.094), even though a larger proportion of individuals with up to 8 years of education or more is observed in the discarded group (91.4%) compared to the confirmed ones (78.4%). A higher proportion of individuals whose disease was discarded were previously vaccinated against YF (14.3%) compared to those who had the disease (3.8%), with no statistical difference (p = 0.105). Hospitalization occurred in 89.7% of confirmed cases, with significant difference (p < 0.001) if compared to the discarded cases (54.3%), as well as evolution to death, which occurred with 8.3% of those discarded and 29.5% of those confirmed, p = 0.016).
We analyzed signs, symptoms, and laboratorial alterations to verify possible predictive characteristics of YF among suspect cases attended. Initially, univariate analysis was performed, comparing confirmed and discarded cases of YF (Table 1). All variables with p < 0.20 were included in the logistic regression model. Two models show the significant and adjustment variables ( Table 2).
The quality of adjustment of the multivariate models were evaluated by Hosmer-Lemeshow test, whose p-value was not statistically significant (   Based on such factors, three different case definitions were proposed. a) Proposed definition 1: individual with acute onset of fever (reported or measured), followed by elevation of AST superior to twice the reference value AND leukopenia AND thrombocytopenia.
b) Proposed definition 2: individual with acute onset of fever (reported or measured), followed by elevation of ALT superior to twice the reference value AND leukopenia AND thrombocytopenia.
c) Proposed definition 3: individual with acute onset of fever (reported or measured), followed by elevation of ALT or AST superior to twice the reference value AND leukopenia OR thrombocytopenia.
Accuracy analysis was also compared with official definitions from Brazilian Ministry of Health (2017) and from World Health Organization (2015) (

DISCUSSION
Case definition is an important tool for epidemiological surveillance, allowing detection of cases, estimation of incidence, and identification of epidemics. Standardization of case definition also contributes to evaluation of effectiveness of control measures and incidence comparison in different regions and periods. Case definition must be simple and useful, combining clinical, laboratorial, and epidemiological characteristics, depending on the monitoring objectives for each disease or health condition. A definition with high sensitivity and specificity is desirable, but these attributes must be balanced, considering the increase of sensitivity of great importance when extension of an epidemic needs to be assessed 10 . Balance must be reached between the need of high sensitivity at the expense of screening false-positive cases 11 .
Brazilian Ministry of Health and World Health Organization's case definitions presented lower sensitivity, specificity, PVN, and accuracy, with moderate PVP. YF in Brazil presents low incidence, with significant increase of cases in 2017 and 2018, especially in the Southeastern region. A more sensitive case definition is necessary for diseases with low incidence, thus reducing the number of false negatives, considering the severity of disease and the possibility of prevention 12 .
Predictive factors of YF in the analyzed population were transaminases greater than twice the reference value, leukopenia, and thrombocytopenia. A study 13 analyzed risk factors for death in 72 confirmed cases of YF and it identified, among recovered cases, the presence of leukopenia and increase of transaminases and thrombocytopenia. The analysis of regression showed independent factors related to death the increase of transaminases, age, and creatinine increase 13 . Another study, which analyzed 76 cases of YF, showed leukopenia, thrombocytopenia, and increase of transaminases among the cases. When risk factors for death were analyzed, there was statistical significance for the increase of transaminases, neutrophilia, and viral load 14 . Analysis of 52 YF confirmed cases showed increase of transaminases and thrombocytopenia. However, logistic regression showed that alteration of renal excretion and increase of transaminases were significantly associated to death 15  In this proposed definition, the presence of fever was followed by elevation of ALT or AST greater than twice the reference value and leukopenia or thrombocytopenia. Considering the epidemiological situation with high or low case incidence, we must adapt the case definition regarding sensitivity and specificity. In a scenario of low incidence of disease, a higher sensitivity is desirable, even if it increases the number of false positives, to minimize the number of non-detected cases. However, a surveillance system with high PVP, low false positive reports, would not lead to wasted resources on cases that do not actually exist 11 . Therefore, definitions proposed by Brazilian Ministry of Health and WHO presented low sensitivity, thus requiring more sensitive definitions to detect most of YF cases. Other studies developed definitions with the use of similar methodology 17 . Incorporation of laboratorial results made the definition more sensitive, since transaminases alterations in serum are earlier identified than clinical manifestation of jaundice, a signal included in both case definitions (Brazil and WHO) that may be absent in some cases 18 .
Limitation of study may be attributed to the use of data from one hospital only, consequently with a small sample size that can lead to inaccuracies in the association measures. However, Instituto de Infectologia Emílio Ribas is a reference public hospital for infectious disease in the state of São Paulo. Furthermore, cases were identified during a YF epidemic in some regions of the state. Thus, increasing the chance of virus detection in investigated cases. Most cases confirmed the YF virus detection by real-time polymerase chain reaction (RT-PCR) technique (91%). Only seven cases were confirmed by serology, an exam that can crossreact with other flaviviruses. However, four of these cases had jaundice, one associated with hemorrhage that progressed to death. These cases were from regions with yellow fever transmission in the same period. The use of laboratorial exams to case definition can be difficult in regions with lack of access to laboratory diagnosis. Nevertheless, the inclusion of laboratorial criteria, associated to symptomatology, increased sensitivity of case definition, making it useful in situations of outbreak and in sentinel investigation of feverish syndromes for differential diagnosis, especially considering coexistence of many arboviruses in Brazil with similar symptomatology. Furthermore, detection of YF virus in densely populated peri-urban regions with a high rate of Aedes aegypti infestation increases the risk of urban YF epidemic. Therefore, case definitions with high accuracy allow implementation of more timely preventive measures, such as vaccination of population under risk.